The following are publications disclosing background information related to the present invention: G. A. Held et al. (1982) Proc. Natl. Acad. Sci. USA 77:6065-6069; A. Klier et al. (1982) EMBO J. 1:791-799; A. Klier et al. (1983) Nucl. Acids Res. 11:3973-3987; H. E. Schnepf and H. R. Whitely (1981) Proc. Natl. Acad. Sci. USA 78:2893-2897; H. E. Schnepf and X. R. Whitely, European Pat. application 63,949; H. R. Whitely et al. (1982) Molecular Cloning and Gene Regulation in Bacilli, eds: A. T. Ganesan et al., pp. 131-144; H. C. Wong et al. (1983) J. Biol. Chem. 258:1960-1967. R. M. Faust et al. (1974) J. Invertebr. Pathol. 24:365-373, T. Yamamoto and R. E. McLaughlin (1981) Biochem. Biophys. Res. Commun. 103:414-421, and H. E. Huber and P. Luthy (1981) in Pathogenesis of Invertebrate Microbiol. Diseases, ed.: E. W. Davidson, pp. 209-234, report production of activated toxin from crystal protein protoxin. None of the above publications report that partial protoxin genes when transcribed and translated produced insecticidal proteins as disclosed herein. These publications are discussed in the Background section on Molecular Biology. S. Chang (1983) Trends Biotechnol. 1:100-101, reported that the DNA sequence of the HD-1 gene had been publicly presented, (ref. 5 therein), and that the HD-1 toxin moiety resides in the amino-terminal 68kD peptide. M. J. Adang and J. D. Kemp, U.S. patent application Ser. No. 535,354, which is hereby incorporated by reference, described a plasmid, p123/58-10 therein, pBt73-10 herein, containing a partial protexin gene that, when transformed into E. coli, directed synthesis of an Insecticidal protein. M. J. Adang and J. D. Kemp, supra, and R. F. Barker and J. D. Kemp, U.S. patent application Ser. No. 553,786, which is hereby incorporated by reference, both teach expression of the same pBt73-10 partial protoxin structural gene in plants cells. Detailed comparisons of results disclosed as part of the present application with published reports are also detailed herein in the Examples, especially Example 5.
Chemistry
Bacillus thuringiensis, a species of bacteria closely related to B. cereus, forms a proteinacious crystalline inclusion during sporulation. This crystal is parasporal, forming within the cell at the end opposite from the developing spore. The crystal protein, often referred to as the .delta.-endotoxin, has two forms: a nontoxic protoxin of approximate molecular weight (MW) of 130 kilodaltons (kD), and a toxin having an approx. MW of 68 kD. The crystal contains the protoxin protein which is activated in the gut of larvae of a number of insect species. M. J. Klowden et al. (1983) Appl. Envir. Microbiol. 46:312-315, have shown solubilized protoxin from B. thuringiensis var. israelensis is toxic to Aedes aegypti adults. A 65kD "mosquito toxin" seems to be isolatable without an activation step from crystals of HD-1 (T. Yamamoto and R. E. McLaughlin (1981) Biochem. Biophys. Res. Commun. 103:414-421). During activation, the protoxin is cleaved into two polypeptides, one or both of which are toxic. In vivo, the crystal is activated by being solubilized and converted to toxic form by the alkalinity and proteases of the insect gut.
In vitro the protoxin may be solubilized by extremely high pH (e.g. pH 12), by reducing agents under moderately basic conditions (e.g. pH 10), or by strong denaturants (guanidium, urea) under neutral conditions (pH 7). Once solubilized, the crystal protein may be activated in vitro by the action of the protease such as trypsin (R. M. Faust et al. (1974) J. Invertebr. Pathol. 24:365-373). Activation of the protoxin has been reviewed by H. E. Huber and P. Luthy (1981) in Pathogenesis of Invertebrate Microbiol. Diseases, ed.: E. W. Davidson, pp. 209-234. The crystal protein is reported to be antigenically related to proteins within both the spore coat and the vegetative cell wall. Carbohydrate is not involved in the toxic properties of the protein.
Toxicology
B. thuringiensis and its crystalline end in are useful because the crystal protein is an insecticidal protein known to be poisonous to the larvae of over a hundred of species of insects, most commonly those from the orders Lepidoptera and Diptera. Insects susceptible to the action of the B. thuringiensis crystal protein include, but need not be limited to, those listed in Table 1. Many of these insect species are economically important pests. Plants which can be protected by application of the crystal protein include, but need not be limited to, those listed in Table 2. Different varieties of B. thuringiensis, which include, but need not be limited to, those listed in Table 3, have different host ranges (R. M. Faust et al. (1982) in Genetic Engineering in the Plant Sciences, ed. N. J. Panapolous, pp. 225-254); this probably reflects the toxicity of a given crystal protein in a particular host. The crystal protein is highly specific to insects; in over two decades of commercial application of sporulated B. thuringiensis cells to crops and ornamentals there has been no known case of effects to plants or noninsect animals. The efficacy and safety of the endotoxin have been reviewed by R. M. Faust et al., supra. Other useful reviews include those by P. G. Fast (1981) in Microbial Control of Pests and Plant Diseases, 1970-1980, ed.: H. D. Burges, pp. 223-248, and H. E. Huber and P. Luthy (1981) in Pathogenesis of Invertebrate Microbial Diseases, ed.: E. W. Davidson, pp. 209-234.
Molecular Biology
The crystal protein gene usually can be found on one of several large plasmids that have been found in Bacillus thuringiensis, though in some strains it may be located on the chromosome (J. W. Kronstad et al. (1983) J. Bacteriol. 154:419-428; J. M. Gonzalez Jr. et al. (1981) Plasmid 5:31-365). Crystal protein genes have been cloned into plasmids that can grow in E. coli by several laboratories.
Whiteley's group (H. R. Whiteley et al. (1982) in Molecular Cloning and Gene Regulation in Bacilli, eds.: A. T. Ganesan et al., pp. 131-144, H. E. Schnepf and H. R. Whiteley (1981) Proc. Natl. Acad. Sci. USA 78:2893-2897, and European Pat. application 63,949) reported the cloning of the protoxin gene from B. thuringiensis var. kurstaki strains HD-1-Dipel and HD-73, using the enzymes Sau3AI (under partial digest conditions) and BglII, respectively, to insert large gene-bearing fragments having approximate sizes of 12 kbp and 16 kbp into the BamHI site of the E. coli plasmid vector pBR322. The HD-1 crystal protein gene was observed to be contained within a 6.6 kilobase pair (kbp) HindIII fragment. Crystal protein which was toxic to larvae, immunologically identifiable, and the same size as authentic protoxin, was observed to be produced by transformed E. coli cells containing pBR322 derivatives having such large DNA segments containing the HD-1-Dipel gene or subclones of that gene. This indicated that the Bacillus gene was transcribed, probably from its own promoter, and translated in E. coli. Additionally, this finding suggested that the toxic activity of the protein product is independent of the location of its synthesis. That the gene was expressed when the fragment containing it was inserted into the vector in either orientation suggests that transcription was controlled by its own promoter. Whiteley et al., supra, reported a construction deleting the 3'-end of the HD-1 toxin coding sequences along with the nontoxin coding sequence of the protoxin. The transcriptional and translational start sites, as well as the deduced sequence for the amino-terminal 333 amino acids of the HD-1-Dipel protoxin, have been determined by nucleic acid sequencing (H. C. Wong et al. (1983) J. Biol. Chem. 258:1960-1967). The insecticidal gene was found to have the expected bacterial ribosome binding and translational start (ATG) sites along with commonly found sequences at -10 and -35 (relative to the 5'-end of the mRNA) that are involved in initiation of transcription in bacteria such as B. subtilis. Wong et al., supra localized the HD-1 crystal protein gene by transposon mutagenesis, noted that transposon insertion in the 3'-end of the gene could result in production in E. coli of 68kD peptides, but did not report any insecticidal activity to be associated with extracts of strains that produce 68kD peptides while lacking 130 kD protoxin.
A. Klier et al. (1982) EMBO J. 1:791-799, have reported the cloning of the crystal protein-gene from B. thuringiensis strain berliner 1715 in pBR322. Using the enzyme BamHI, a large 14 kbp fragment carrying the crystal protein gene was moved into the vector pHV33, which can replicate in both E. coli and Bacillus. In both E. coli and sporulating B. subtilis, the pHV33-based clone directed the synthesis of full-size (130 kD) protoxin which formed cytoplasmic inclusion bodies and reacted with antibodies prepared against authentic protein. Extracts of E. coli cells harboring the pBR322 or pHV33-based plasmids were toxic to larvae. In further work, A. Klier et al. (1983) Nucleic Acids Res. 11:3973-3987, have transcribed the berliner crystal protein gene in vitro and have reported on the sequence of the promoter region, together with the first 11 codons of the crystal protein. The bacterial ribosome binding and translational start sites were identified. Though the expected "-10" sequence was identified, no homology to other promoters has yet been seen near -35.
G. A. Held et al. (1982) Proc. Natl. Acad. Sci. USA 77:6065-6069 reported the cloning of a crystal protein gene from the variety kurstaki in a phage X-based cloning vector Charon4A. E. coli cells infected with one of the Charon clones produced antigen that was the same size as the protoxin (130 kD) and was toxic to larvae. A 4.6 kbp EcoRI fragment of this Charon clone was moved into pHV33- and an E. coli plasmid vector, pBR328. Again, 130 kD antigenically identifiable crystal protein was produced by both E. coli and B. subtilis strains harboring the appropriate plasmids. A B. thuringiensis chromosomal sequence which cross-hybridized with the cloned crystal protein gene was identified in B. thuringiensis strains which do not produce crystal protein during sporulation.